Identification of (2S,3S)-β-Methyltryptophan as the Real Biosynthetic Intermediate of Antitumor Agent Streptonigrin.

Dekun Kong,Fei Xu,Liping Zhang,Zhang Zhang,Nelson L Brock,Zixin Deng,Yi Zou,Shuangjun Lin

doi:10.1038/srep20273

Abstract

Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on (2S,3R)-β-methyltryptophan as an intermediate. In this study, the biosynthesis of (2S,3R)-β-methyltryptophan and its isomer (2S,3S)-β-methyltryptophan by enzymes from the streptonigrin biosynthetic pathway is demonstrated. StnR is a pyridoxal 5′-phosphate (PLP)-dependent aminotransferase that catalyzes a transamination between L-tryptophan and β-methyl indolepyruvate. StnQ1 is an S-adenosylmethionine (SAM)-dependent C-methyltransferase and catalyzes β-methylation of indolepyruvate to generate (R)-β-methyl indolepyruvate. Although StnR exhibited a significant preference for (S)-β-methyl indolepyruvate over the (R)-epimer, StnQ1 and StnR together catalyze (2S,3R)-β-methyltryptophan formation from L-tryptophan. StnK3 is a cupin superfamily protein responsible for conversion of (R)-β-methyl indolepyruvate to its (S)-epimer and enables (2S,3S)-β-methyltryptophan biosynthesis from L-tryptophan when combined with StnQ1 and StnR. Most importantly, (2S,3S)-β-methyltryptophan was established as the biosynthetic intermediate of the streptonigrin pathway by feeding experiments with a knockout mutant, contradicting the previous proposal that stated (2S,3R)-β-methyltryptophan as the intermediate. These data set the stage for the complete elucidation of the streptonigrin biosynthetic pathway, which would unlock the potential of creating new streptonigrin analogues by genetic manipulation of the biosynthetic machinery.

Highlights

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid produced by Streptomyces flocculus[1,2]
In order to characterize the enzymes of this cassette in vitro, the three genes were individually overexpressed in E. coli BL21(DE3) and the corresponding N-terminally His6-tagged proteins were purified to near homogeneity (Figure S2)
It has been shown that the aminotransferase StnR is specific for l-tryptophan as an amino donor, and preferentially catalyzes the transamination of the amino acceptor (S)-β -methyl indolepyruvate over the epimer (R)-β -methyl indolepyruvate

Summary

Introduction

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid produced by Streptomyces flocculus[1,2]. Initial biosynthetic studies of STN in S. flocculus suggested that the 4-phenyl picolinic acid moiety is derived from l-tryptophan via β -methyltryptophan as an intermediate[26]. The biosynthesis of (2S,3R)- and (2S,3S)-β -MeTrp by enzymes from the biosynthetic pathway of STN was characterized in vitro. Bioinformatic analysis of the stn gene cluster revealed a three-gene cassette (stnQ1-stnK3-stnR) encoding an S-adenosylmethionine (SAM)-dependent C-methyltransferase, a cupin-fold epimerase, and a pyridoxal 5′ -phosphate (PLP)-dependent aminotransferase, respectively. This gene cassette is homologous to the marI-marH-marG cassette that generates (2S,3S)-β -MeTrp for the biosynthetic pathway of maremycins (Figure S1; identity/similarity: 64%/77% for StnQ1/MarI, 82%/86% for StnK3/MarH, and 64%/73% for StnR/ MarG)[34]. The bioinformatic data suggests that the stnQ1-stnK3-stnR cassette is responsible for the biosynthesis of (2S,3S)-β -MeTrp as well

Methods

Results

Conclusion